As stated above, the present invention relates generally to the field of the transgenic production of fusion proteins in the milk of transgenic animals. More particularly, it concerns improved methods for generating transgenic non-human mammalian animals, of various species, capable of producing a variety of fusion proteins of interest.
Currently, there are numerous polypeptides, macromolecules and/or proteins (“proteins of interest”) possessing one or more potential therapeutic activities cannot be exploited pharmaceutically. There may be various reasons for this inability, such as low stability in vivo, altered glycosylation patterns found in proteins from non-eukaryotic cells, improper translational processing, tertiary structure, fragile structure, immunogenicity, the difficulty of producing them on an industrially acceptable scale or the like. Moreover, some therapeutically interesting proteins do not give the expected results in vivo because of problems related to the method of their purification, administration or pharmacokinetics.
The present invention makes it possible to overcome these disadvantages. The instant invention provides new fusion molecules which permits the exploitation of the physiological properties or effects of the proteins of interest. The present invention results especially from the demonstration that it is possible to fuse a physiologically active sequence derived from a biologically active protein to another recombinant protein structure consisting of a protein sequence retaining the physiological activity of human alpha-fetoprotein to derive a bi-functional fusion protein, without impairing the biological properties of either the alpha-fetoprotein or the second protein moiety thereof. It also results from the demonstration by the Inventors that the recombinant human alpha-fetoprotein protein sequence of the invention (“AFP”) can improve the half-life of the fusion proteins of the invention, and add synergistic therapeutic efficacy as well.
The physiological effects of AFP have been shown in the prior art to include both stimulative and inhibitory effects on various cell types. These effects in large part are determined by the target cell type, the relative concentration of AFP, and the presence of other cytokines and growth factors. For example, AFP can inhibit the growth of many types of tumor cells, and, in particular, inhibits estrogen-stimulated or estrogen-sensitive cell growth. Conversely, AFP stimulates the growth of normal embryonic fibroblasts. AFP has also been shown to have both immunosuppressive and immunoproliferative effects. Therefore the therapeutic effectiveness of an AFP fusion protein can be utilized in such a way as to maximize the pharmacologic effectiveness of treatments for various disease states, especially using bi-functional molecules.
The fusion proteins according to the current invention make it possible to maintain, in the body of an animal, a desirable biological activity for a prolonged period. The proteins of interest according to the current invention can also be expressed secreted by recombinant organisms, such as in cell culture production facilities, or transgenic mammals, at levels permitting their commercial exploitation. Along this line, transgenic mammals are a preferred manufacturing and expression vehicle for the fusion proteins of the invention.
Mammals having certain desired traits or characteristics, such as increased weight, milk content, milk production volume, length of lactation interval and disease resistance have long been desired. Traditional breeding processes are capable of producing animals with some specifically desired traits, but often these traits these are often accompanied by a number of undesired characteristics, and are often too time-consuming, costly and unreliable to develop. Moreover, these processes are completely incapable of allowing a specific animal line from producing gene products, such as desirable protein therapeutics that are otherwise entirely absent from the genetic complement of the species in question (i.e., human or humanized plasma protein or other molecules in ungulate milk). The development of technology capable of generating transgenic animals provides a means for exceptional precision in the production of animals that are engineered to carry specific traits or are designed to express certain proteins or other novel molecular compounds of therapeutic, scientific or commercial value. That is, transgenic animals are animals that carry the gene(s) of interest that has been deliberately introduced into existing somatic cells and/or germ line cells at an early stage of development. As the animals develop and grow the protein product or specific developmental change engineered into the animal is expressed, and at that point is present in the genetic complement of that animal and its offspring.
In a preferred embodiment the current invention provides for the bulk production of a bi-functional fusion protein of interest in the milk of transgenic mammals. The production of a fusion protein of interest in milk is ideal as a bulk process because very large volumes of milk that can be produced, collected and purified using known dairy technology. A second advantage of using a transgenic mammalian process is that some reactions which can be essential for biological activity in humans, for example carboxy-terminal amidation, are difficult to perform in good yield by currently available chemical means or in bacterial or other in vitro situations. For example, carboxy-terminal amidation is catalyzed by a specific enzyme which recognizes and modifies a fusion protein of interest or proteins with a glycine residue at the carboxy terminus. Therefore, suitably designed fusion proteins of the invention can be specifically amidated before secretion into the milk of transgenic animals. This is only one example of a range of post-translational modifications which can be carried out by the biosynthetic pathways in the mammary gland and which can potentially be harnessed for the synthesis of particular fusion proteins. Other examples of desirable post-translational modifications include di-sulfide bridge formation, γ-carboxylation of glutamic acid residues and the addition of O- and N- linked glycosylation.
With regard to a physiologically active fragment of AFP it should also be noted that the non-glycosylated form of recombinant AFP exhibits similar biological properties to the normally glycosylated form and provides a standardized consistent product due to the lack of glycosylation variability. It may also be more easily produced in in vitro or transgenic systems. Therefore, non-glycosylated AFP is a preferred form for commercial production.
According to the prior art, the generation of an animal capable of producing a recombinant protein of interest is known. However, what remained unknown prior to the current invention was the level of genetic manipulation required for the current invention, the modified sequences of the various fusion protein components available, the synergistic effect of the current bi-functional molecules, and the disease states or pathologies in which they are useful.
Accordingly, a need exists for improved methods of therapeutic composition generation. The methods of the invention are typically applied to primary somatic cells, in the context of nuclear transfer, for the generation of transgenic animals useful in the production of recombinant fusion bi-functional proteins of interest in their milk.